The immune system contains a system of dendritic cells that is specialized to present antigens and initiate several T-dependent immune responses. Dendritic cells are distributed widely throughout the body in various tissues. The distribution of dendritic cells has been reviewed in (1). Dendritic cells are found in nonlymphoid organs either close to body surfaces, as in the skin and airways, or in interstitial regions of organs like heart and liver. Dendritic cells, possibly under the control of the cytokine granulocyte macrophage colony-stimulating factor, (hereinafter GM-CSF), can undergo a maturation process that does not entail cell proliferation (2,3). Initially, the dendritic cells process and present antigens most likely on abundant, newly synthesized MHC class II molecules, and then strong accessory and cell-cell adhesion functions are acquired (4-7). Dendritic cells can migrate via the blood and lymph to lymphoid organs (8-10). There, presumably as the "interdigitating" cells of the T-area (8,11-13), antigens can be presented to T cells in the recirculating pool (14). However, little is known about the progenitors of dendritic cells in the different compartments outlined above.
The efficacy of dendritic cells in delivering antigens in such a way that a strong immune response ensues i.e., "immunogenicity", is widely acknowledged, but the use of these cells is hampered by the fact that there are very few in any given organ. In human blood, for example, about 0.1% of the white cells are dendritic cells (25) and these have not been induced to grow until this time. Similarly, previous studies (20, 21) have not reported the development, in culture, of large numbers of dendritic cells from bone marrow. A more recent report described the development of dendritic cells in GM-CSF supplemented marrow cultures, however no documentation as to the origin of the dendritic cells or use of proliferating aggregates as an enriched source of dendritic cells was observed. (Scheicher et al. (1992)) J. Immunol. Method. 154:253-264. While dendritic cells can process foreign antigens into peptides that immunologically active T cells must recognize (4,6,7,14) i.e., dendritic cells accomplish the phenomenon of "antigen presentation", the low numbers of dendritic cells prohibits their use in identifying immunogenic peptides.
Dendritic cells in spleen (15) and afferent lymph (16,17) are not in the cell cycle but arise from a proliferating precursor. Ultimately, dendritic cells emanate from the bone marrow (15,16,18,19), yet it has been difficult to generate these cells in culture except for two reports describing their formation in small numbers (20,21). Although a bone marrow precursor cell has been reported, conditions have not been reported that direct its proliferation in culture (Steinman, R. (1991)) "The Dendritic Cell System and Its Role In Immunogenicity", Ann. Rev. Immunol., 9:271-96. Identification of proliferating dendritic cells in bone marrow, in contrast to blood, is difficult because there are large numbers of granulocytes that develop in response to GM-CSF and these crowd the immature dendritic cell cultures, preventing maturation of the dendritic precursors. The use of cell surface markers to enrich bone marrow dendritic cell precursors has been reported to result in only modest increases because the markers are also expressed by numerous non-dendritic bone marrow cells (Bowers, W. E. and Goodell (1989)), "Dendritic Cell Ontogeny" Res. Immunol. 140:880-883.
Relatively small numbers of dendritic cells have also been isolated from blood (Vakkila J. et al. (1990) "Human Peripheral blood-derived dendritic cells do not produce interleukin 1.alpha., interleukin 1.beta., or interleukin 6" Scand. J. Immunol. 31:345-352; Van Voorhis W. C. et al., (1982) "Human Dendritic Cells", J.Exp. Med., 1172-1187.) However, the presence in blood of dendritic cell precursors has not been reported and as recently as 1989 the relationship between blood dendritic cells and mature dendritic cells in other tissues was uncertain. Furthermore, it was recognized that dendritic cells are "rare and difficult to isolate and have not as yet been shown to give rise to DC [dendritic cells] in peripheral tissues." (MacPherson G. G. (1989) "Lymphoid Dendritic cells: Their life history and roles in immune responses", Res. Immunol. 140:877-926).
Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a factor which modulates the maturation and function of dendritic cells. (Witmer-Pack et al, (1987) "Granulocyte/macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells". J.Exp.Med. 166:1484-1498; Heufler C. et al., (1988) "Granulocyte/macrophage colony-stimulating factor and interleukin 1 mediate the maturation of murine epidermal Langerhans cells into potent immunostimulatory dendritic cells", J. Exp. Med. 167:700-705). GM-CSF stimulated maturation of dendritic cells in vitro suggests that the presence of GM-CSF in a culture of dendritic cell precursors would mediate maturation into immunologically active cells, but the important goal of achieving extensive dendritic cell growth has yet to be solved.
T-dependent immune responses are characterized by the activation of T-helper cells in the production of antibody by B cells. An advantage of T-dependent over T-independent immune responses is that the T-dependent responses have memory, i.e. cells remain primed to respond to antigen with rapid production of antibody even in the absence of antigen and the immune response is therefore "boostable". T-independent immune responses are, in contrast, relatively poor in children and lack a booster response when a T-independent antigen is repeatedly administered. The immunologic memory of T cells likely reflects two consequences of the first, "primary" or "sensitizing" limb of the immune response: (a) an expanded number of antigen-specific T cells that grow in response to antigen-bearing dendritic cells, and (b) the enhanced functional properties of individual T cells that occurs after dendritic cell priming (Inaba et al., (1984) Resting and sensitized T lymphocytes exhibit distinct stimulatory (antigen presenting cell) requirements for growth and lymphokine release; J.Exp.Med. 160:868-876; Inaba and Steinman, (1985) "Protein-specific helper T lymphocyte formation initiated by dendritic cells", Science 229: 475-479; Inaba et al., (1985) "Properties of memory T lymphocytes isolated from the mixed leukocyte reaction", Proc.Natl.Acad.Sci. 82:7686-7690).
Certain types of antigens characteristically elicit T-cell dependent antibody responses whereas others elicit a T-cell independent response. For example, polysaccharides generally elicit a T-cell independent immune response. There is no memory response and therefore no protection to subsequent infection with the polysaccharide antigen. Proteins, however, do elicit a T-cell dependent response in infants. The development of conjugate vaccines containing a polysaccharide covalently coupled to a protein converts the polysaccharide T-independent response to a T-dependent response. Unfortunately, little is known concerning the sites on proteins which confer their T-cell dependent character, therefore hampering the design of more specific immunogens.
As stated above, dendritic cells play a crucial role in the initiation of T-cell dependent responses. Dendritic cells bind and modify antigens in a manner such that the modified antigen when presented on the surface of the dendritic cell can activate T-cells to participate in the eventual production of antibodies. The modification of antigens by dendritic cells may, for example, include fragmenting a protein to produce peptides which have regions which specifically are capable of activating T-cells.
The events whereby cells fragment antigens into peptides, and then present these peptides in association with products of the major histocompatibility complex, (MHC) are termed "antigen presentation". The MHC is a region of highly polymorphic genes whose products are expressed on the surfaces of a variety of cells. MHC antigens are the principal determinants of graft rejection. Two different types of MHC gene products, class I and class II MHC molecules, have been identified. T cells recognize foreign antigens bound to only one specific class I or class II MHC molecule. The patterns of antigen association with class I or class II MHC molecules determine which T cells are stimulated. For instance, peptide fragments derived from extra cellular proteins usually bind to class II MHC molecules, whereas proteins endogenously transcribed in dendritic cells generally associate with newly synthesized class I MHC molecules. As a consequence, exogenously and endogenously synthesized proteins are typically recognized by distinct T cell populations.
Dendritic cells are specialized antigen presenting cells in the immune response of whole animals (14,31). Again however, the ability to use dendritic cells to identify and extract the immunogenic peptides is hampered by the small numbers of these specialized antigen presenting cells.
Particle uptake is a specialized activity of mononuclear and polymorphonuclear phagocytes. Dead cells, immune complexes, and microorganisms all are avidly internalized. Following fusion with hydrolase-rich lysosomes, the ingested particles are degraded (60,61). This degradation must be to the level of permeable amino acids (62,63) and saccharides, otherwise the vacuolar apparatus would swell with indigestible materials (64,65). Such clearance and digestive functions of phagocytes contribute to wound healing, tissue remodeling, and host defense.
Another consequence of endocytosis, the processing of antigens by antigen presenting cells (APCs), differs in many respects from the scavenging function of phagocytosis. First, processing requires the generation of peptides at least 8-18 amino acids in length (66,67), while scavenging entails digestion to amino acids (62,63). Secondly, presentation requires the binding of peptides to MHC class II products (6,68), whereas scavenging does not require MHC products. Thirdly, antigen presentation can function at a low capacity, since only a few hundred molecules of ligand need to be generated for successful stimulation of certain T-T hybrids (69,70) and primary T cell populations (71). During scavenging, phagocytes readily clear and destroy hundreds of thousands of protein molecules each hour (63). Lastly, antigen presentation is best carried out by cells that are rich in MHC class II but show little phagocytic activity and few lysosomes, i.e., dendritic cells and B cells, while phagocytes (macrophages and neutrophils) often have low levels of class II and abundant lysosomes. These observations, together with the identification of antigenic specializations within the endocytic system of dendritic cells and B cells, have lead to the suggestion that the machinery required for antigen presentation may differ from that required for scavenging, both quantitatively and qualitatively (31).
In the case of dendritic cells, there have been indications that these APCs are at some point during their lifetime capable of phagocytic activity. Pugh et al. noted Feulgen-stained inclusions in some afferent lymph dendritic cells and suggested that phagocytosis of other cells had taken place prior to entry into the lymph (16). Fossum noted phagocytic inclusions in the interdigitating dendritic cells of the T cell areas in mice that were rejecting allogeneic leukocytes (71). Reis e Sousa et al. (74) found that freshly isolated epidermal Langerhans cells, which are immature but nonproliferating dendritic cells, internalize small amounts of certain particulates. Neither report, however, demonstrates or suggests the occurrence of phagocytosis when particles are administered to cultures of proliferating dendritic cells.
Injection of dendritic cells pulsed with pathogenic lymphocytes into mammals to elicit an active immune response against lymphoma is the subject of PCT patent application WO 91/13632. In addition, Francotte and Urbain, Proc. Nat'l. Acad. Sci., USA 82:8149 (1985) reported that mouse dendritic cells, pulsed in vitro with virus and injected back into mice, enhances the primary response and the secondary response to the virus. Neither the report by Francotte and Urbain and patent application WO 91/13632 provide a practical method of using dendritic cells as an adjuvant to activate the immune response because both of these methods depend on dendritic cells obtained from spleen, an impractical source of cells for most therapies or immunization procedures. In addition, neither report provides a method to obtain dendritic cells in sufficient quantities to be clinically useful.